Ketohexokinase CRISPR Activation Plasmid (h): sc-403399-ACT

Human KHK encodes ketohexokinase, a liver- and kidney-enriched fructokinase that catalyzes phosphorylation of fructose to fructose-1-phosphate, initiating fructose metabolism and coupling carbohydrate flux to downstream glycolytic and lipogenic pathways. By regulating cellular handling of dietary fructose, KHK influences ATP utilization, phosphate homeostasis, and metabolite partitioning into triglyceride synthesis and uric acid–linked purine turnover. Altered KHK activity has been associated with inborn errors of fructose metabolism and with metabolic phenotypes involving hepatic lipid accumulation and insulin resistance–related signaling. As a node in fructose-driven carbon metabolism, KHK is widely studied in nutrient sensing, hepatocyte metabolic reprogramming, and stress responses linked to high-fructose exposure.

Ketohexokinase CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous KHK expression without altering the underlying DNA sequence.

Ketohexokinase CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the KHK locus in human cell lines. The system is built around a catalytically inactive Cas9 (dCas9) carrying two inactivating mutations (D10A and N863A) that eliminate nuclease activity while preserving DNA binding. This dCas9 is fused to VP64, a potent transcriptional activator, and is co-expressed with a blasticidin resistance gene for selection. The second plasmid encodes the MS2-p65-HSF1 fusion protein, a secondary activator complex that works in concert with dCas9-VP64, alongside a hygromycin resistance gene. The third plasmid encodes a target-specific 20 nt sgRNA fused to two MS2 RNA aptamers that recruit the MS2-p65-HSF1 complex to the activation site, accompanied by a puromycin resistance gene. The three plasmids are delivered at a 1:1:1 mass ratio for balanced expression of all system components.

Once assembled at the target locus, the SAM complex binds within approximately 200 bp upstream of the KHK transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Ketohexokinase expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native KHK locus and enabling the study of Ketohexokinase-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Ketohexokinase pathway restoration in tumor cells with silenced or reduced KHK expression.

For Research Use Only. Not Intended for Diagnostic or Therapeutic Use.